A D-pathway mutation decouples the Paracoccus denitrificans cytochrome c oxidase by altering the side-chain orientation of a distant conserved glutamate.
Asparagine 131, located near the cytoplasmic entrance of the D-pathway in subunit I of the Paracoccus denitrificans aa(3) cytochrome c oxidase, is a residue crucial for proton pumping. When replaced by an aspartate, the mutant enzyme is completely decoupled: while retaining full cytochrome c oxidation activity, it does not pump protons. The same phenotype is observed for two other substitutions at this position (N131E and N131C), whereas a conservative replacement by glutamine affects both activities of the enzyme. The N131D variant oxidase was crystallized and its structure was solved to 2.32-A resolution, revealing no significant overall change in the protein structure when compared with the wild type (WT), except for an alternative orientation of the E278 side chain in addition to its WT conformation. Moreover, remarkable differences in the crystallographically resolved chain of water molecules in the D-pathway are found for the variant: four water molecules that are observed in the water chain between N131 and E278 in the WT structure are not visible in the variant, indicating a higher mobility of these water molecules. Electrochemically induced Fourier transform infrared difference spectra of decoupled mutants confirm that the protonation state of E278 is unaltered by these mutations but indicate a distinct perturbation in the hydrogen-bonding environment of this residue. Furthermore, they suggest that the carboxylate side chain of the N131D mutant is deprotonated. These findings are discussed in terms of their mechanistic implications for proton routing through the D-pathway of cytochrome c oxidase.
Dürr
Katharina L
KL
Department of Biochemistry, Molecular Genetics Group, Johann Wolfgang Goethe University, Frankfurt/Main, Germany. Katharina.Duerr@TU-Berlin.de
Koepke
Juergen
J
Hellwig
Petra
P
Müller
Hannelore
H
Angerer
Heike
H
Peng
Guohong
G
Olkhova
Elena
E
Richter
Oliver-Matthias H
OM
Ludwig
Bernd
B
Michel
Hartmut
H
D018360:Crystallography, X-Ray; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry; D008958:Models, Molecular; D020125:Mutation, Missense; D010231:Paracoccus denitrificans / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D017494:Proton Pumps / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared
D017494:Proton Pumps; D018698:Glutamic Acid; D003576:Electron Transport Complex IV